Recent events in the United Kingdom have demonstrated very clearly that foot and mouth disease virus (FMDV) is so highly contagious that rapid diagnosis is required to control its spread. See, e.g., Adam, D., Nature 410:398 (2001) and Enserink, M., Science 291:2298-2300 (2001).
Foot and Mouth Disease Virus (FMDV) is actually a group of closely related viruses, classified as members of the genus Aphthovirus and family Picornaviridae. The genus Aphthovirus has two members, FMDV and Equine Rhinitis A Virus (ERV-1). The second genus member, ERV-1, shares some sequence homology with FMDV, but is not a cause of foot and mouth disease (FMD). ERV-1 is the agent of an equine respiratory disease (horses are not susceptible to FMDV).
There are seven serotypes of FMDV: types A, O, C, Asia 1, Sat 1 (South African Territories), Sat 2, and Sat 3. Serotypes are distinguishable by serotype-specific enzyme linked immunosorbent assays (ELISA).
Because of the range of species affected, the high rate of infectivity, and the fact that FMDV is shed before clinical signs occur, FMD is one of the most feared reportable diseases known in North America. Disease caused by FMDV is devastating to farm animals and can have a major economic impact on countries producing cloven-hoofed animals (cattle, pigs, sheep, goats and camelids) or their products. Clearly, new and more sensitive assays for the detection of this disease are needed.
A variety of methods for the detection of FMDV have been developed. These fall into three general categories: 1) detection of FMDV peptides; 2) detection of FMDV generated antibodies; and 3) detection of FMDV genetic material.
A number of peptides have been identified that are unique to the FMDV and are considered diagnostic for its presence. These include both structural proteins as well as non-structural proteins (see, e.g., Yi et al., U.S. Pat. No. 6,048,538; Saeki et al., U.S. Pat. No. 5,639,601).
In other cases methods have been developed to detect antibodies generated by the infected animal to the FMDV. The ELISA assay is a preferred format (see, e.g., Gilles et al., J. Virological Methods 107(1):89-98 (2003); Mackay et al., J. Virological Methods 97(1-2):33-48 (2001); Bergmann et al., Archives of Virology 145(3):473-489 (2000); and Ferris, N. P., Towards Livestock Disease Diagnosis and Control in the 21st Century, Proceedings of an International Symposium on Diagnosis and Control of Livestock Diseases Using Nuclear and Related Techniques, Vienna, Apr. 7-11, 1997 (1998), Meeting Date 1997, 65-77, International Atomic Energy Agency, Vienna, Austria).
A common and effective method of assay has been the use of primer directed nucleic amplification methods for the amplification of diagnostic portions of the FMDV genome. These methods are based on the isolation of primers or probes that are particularly diagnostic for the presence of the virus. Collins et al. (Biochemical and Biophysical Research Communications 297(2):267-274 (2002)) teach an isothermal method of nucleic acid sequence-based amplification using primers based on a variety of loci in the FMDV genome. One of the most popular methods for detection is the use of a method involving reverse transcription followed by polymerase chain reaction (RT-PCR). As its name implies, the method involves the synthesis of DNA by reverse transcription and then the amplification of DNA by PCR. Callahan et al. (WO 02/095074) use this method for the detection of FMDV using primers derived from highly conserved regions of the 3D coding region of the genome. Reid et al. (J. Virological Methods 105(1):67-80 (2002)) teach a fluorogenic RT-PCR assay using a primer/probe set designed from the internal ribosomal entry site region of the virus genome that was capable of detecting all seven serotypes of the FMDV. The primer-based methods are amenable to a variety of formats and kits (see, e.g., Callahan et al., J. American Veterinary Medical Association 220(11): 1636-1642 (2002).
All of the above methods have been used in the detection of FMDV. However, tests with reliable breadth of specificity for “universal” detection of all strains and increased sensitivity, along with ease and reliability of use, are still needed in an FMDV assay. Additionally, because of the high gene mutation rate in the virus, tests directed to different regions of the genome would be useful. There is a need, therefore, for a highly sensitive assay for FMDV that broadly detects most strains of the virus, is rapid, accurate and easily performed.